| Project/Area Number |
09480185
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Molecular biology
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| Research Institution | Nagoya University |
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Principal Investigator |
MASUMOTO Hiroshi Nagoya University, Graduate School of Science, Assistant Professor, 大学院・理学研究科, 講師 (70229384)
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| Co-Investigator(Kenkyū-buntansha) |
NOZAKI Naohito Kanagawa Dental College, Research Associate, 歯学部, 助手 (70222198)
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| Project Period (FY) |
1997 – 1998
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| Project Status |
Completed (Fiscal Year 1998)
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| Budget Amount *help |
¥13,300,000 (Direct Cost: ¥13,300,000)
Fiscal Year 1998: ¥5,000,000 (Direct Cost: ¥5,000,000)
Fiscal Year 1997: ¥8,300,000 (Direct Cost: ¥8,300,000)
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| Keywords | cetromere / teloreme / alphoid DNA / YAC / HAC / CENP-B |
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| Research Abstract |
In order to define a functional human centromere sequence, an artificial chromosome was constructed as a reproducible DNA molecule. Mammalian telomere repeats and a selectable marker were introduced into yeast artificial chromosomes (YACs) containing alphoid DNA from the human chromosome 21 centromere region in a recombination-deficient yeast host. When these modified YACs were introduced into human cultured cells, a YAC with the alphoid DNA from alpha21-I locus, containing CENP-B boxes at a high frequency and a regular repeat array, efficiently formed artificial chromosomes which were maintained stably in the absence of selection and bound centromere/kinetochore proteins, CENP-A, CENP-B, CENP-C and CENP-E.The artificial chromosomes 1 - 5 Mb in size and composed of multimers of the introduced YAC DNA, aligned at metaphase plates and segregated to opposite poles correctly in anaphase. Extensive cytological analyses strongly suggested that they had not acquired host sequences and were formed in all cases by a de novo mechanism. In contrast, artificial chromosomes were never produced with a modified YAC containing alphoid DNA from the alpha21-II locus which contains no CENP-B boxes and has a less regular sequence arrangement. We conclude that alpha21-I alphoid DNA can induce de novo assembly of active centromere/kinetochore structures on artificial chromosomes.
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